Componential analyzer, drug efficacy analyzer, and analysis method

ABSTRACT

Application of the present invention enables quantification of fractions of candidate pharmaceutical compounds (a parent compound and its metabolites), one excreted to the basolateral (Basal/Basolateral)-side via transporters and by diffusion, one excreted to the lumen (Apical)-side, and one remained in the cells. This enables determination of the total amount of the administered candidate pharmaceutical compounds and the distribution ratio of the fractions. The kinetics of the administered candidate pharmaceutical compounds can be evaluated, thereby enabling in vitro screening of an enormous number of candidate pharmaceutical compounds for drug candidates exhibiting the efficacy. The object of the present invention is to provide an apparatus and method for understanding a total picture of pharmacokinetics in vitro by quantifying a fraction of basolateral (Basal/Basolateral) efflux, a fraction of lumen (Apical)-side excretion, and a fraction remaining in a cell of a drug which has been administered to the cell to determine the distribution ratio of each fraction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/847,092, filed Dec. 19, 2017 which is a continuation of U.S.application Ser. No. 14/914,278, filed Feb. 25, 2016, now U.S. Pat. No.9,880,153, issued Jan. 30, 2018, which is a National Stage ofInternational Patent Application No. PCT/JP2014/081135, filed Nov. 26,2014, which claims the benefit of JP 2013-246906, filed Nov. 29, 2013,the entire disclosures of which are herein expressly incorporated byreference.

TECHNICAL FIELD

The present invention relates to an in vitro evaluation of a totalpicture of pharmacokinetics, including uptake, metabolism, andexcretion, useful for new drug development.

BACKGROUND ART

The process of new drug development necessarily involves conducting aclinical trial (“human clinical trial”), which involves administrationinto the human body and verification of the effect, under the regulationof the Act on Pharmaceuticals and Medical Devices and such humanclinical trials and animal testing entail enormous development costs.The cost of overall new drug development has recently increased withthese development costs being the greatest cause. A major cause of thecost is that insufficient efficacy and toxicity of some drug candidatesare not detected in non-clinical animal experiments in the earlierperiod of the development process and the insufficient efficacy or thetoxicity may be found for the first time in a human clinical trial inthe later period of the development process, failing to avoid uselessdevelopment costs and costs for the human clinical trial.

In this context, it is important to select new drug candidate that havethe efficacy and no toxicity early in order to increase the clinicalsuccess rate and reduce the new drug development cost. Therefore, manypharmaceutical companies desire in vitro evaluation systems allowinggood predictions of pharmacokinetics of administered drugs in the humanbody using human cells in an early stage of drug development, instead ofnarrowing down drug candidates solely depending on animal experiments,which have poor correlations with properties of human cells. However, notechniques have been established to attain and analyze total pictures ofpharmacokinetics, including uptake, metabolism, excretion through bileducts and blood vessels, of administered candidate pharmaceuticalcompounds using cells.

A drug needs to be taken up by the liver and metabolized there into ametabolite or left as an original compound (a parent compound) withoutbeing metabolized, then excreted to the basolateral side, andrecirculated in the bloodstream to the target organ in order to exhibitits efficacy in the body. Therefore, such an in vitro testing systemthat can measure the amount of a recirculating candidate pharmaceuticalcompound excreted to the basolateral side after administration andevaluate the efficacy, which is one of the most important indexes in thenew drug development, could be a very useful pharmacokinetics evaluatingsystem. In addition, such a testing system would provide a total pictureof the pharmacokinetics including the amount excreted from cells intobile duct (the amount of loss), which is the amount excreted into a bileduct and then out of the body as urine or feces, and the amount retainedin the cells and thereby the distribution ratio of such fractions.

Patent Literature 1 and 2 disclose techniques for administering a drugto culture cells and evaluating the amount excreted from bile ducts ofthe cells (the amount of loss). These evaluation methods evaluate theamount of drug loss, which is an amount of a drug that is administered,but excreted into a bile duct without exhibiting toxicity or efficacyand then excreted out of the body as urine or feces.

CITATION LIST Patent Literature

-   Patent Literature 1: JP Patent Publication No. 2012-65659 A-   Patent Literature 2: JP Patent Publication No. 8-503610 A (1996)

SUMMARY OF INVENTION Technical Problem

The methods of the conventional art are methods for evaluating theamount of biliary excretion, which is an amount of a component having noefficacy, that is, a method for evaluating an amount lost from the body.In order to obtain information on a component having efficacy, it isdesired to evaluate directly the amount of the component excreted to thebasolateral side. In the conventional art, however, a method foranalyzing the amount of a component excreted into the basolateral sidewas not established. Furthermore, unlike the systems that can evaluateonly a part of pharmacokinetics, a system that quantifies theadministered drug in fractions, as to where in the cells theadministered drug is excreted and where is the drug is retained, such asone excreted to the basolateral side (the basal/basolateral side), oneexcreted into the lumen side (the apical side), and one retained in thecells, provides a total picture of pharmacokinetics in vitro, therebyenabling a highly accurate evaluation of the efficacy. However, such anevaluation method has not been established in the conventional arteither.

Solution to Problem

For example, provided is a componential analyzer comprising: atemperature controlling unit for controlling temperature in a pluralityof containers; and an analyzing unit for measuring a component in theplurality of containers and analyzing the measured component; whereinthe plurality of containers includes at least a first container and asecond container; the first container and the second container eachcontain a first buffer solution; the temperature controlling unitcontrols temperature in the first container and temperature in thesecond container so that the temperatures are different from each other;and the analyzing unit measures an amount of the component excreted froma cell in the first container to the first buffer solution in the firstcontainer and an amount of the component excreted from a cell in thesecond container to the first buffer solution in the first container,and analyzes an amount of the component excreted via transporters in thecells.

Effects of Invention

Application of the present invention enables direct evaluation ofcomponents, such as drugs, excreted to the basolateral side of cells.Furthermore, quantification of fractions of candidate pharmaceuticalcompounds (a parent compound and its metabolites), one excreted to thebasolateral (basal/basolateral) side by transporters and/or diffusion,one excreted to the lumen (apical) side, and one remained in the cells,and determination of the total amount of the administered candidatepharmaceutical compounds and the distribution ratio of the fractionsenable evaluation of the kinetics of the administered candidatepharmaceutical compounds, thereby improving the accuracy of in vitroscreening of an enormous number of candidate pharmaceutical compoundsfor drug candidates exhibiting the efficacy. As a result, it becomespossible to select candidate pharmaceutical compounds in an early stageand reduce useless animal experiments and unnecessary human clinicaltrials. It contributes to the reduction of attrition rate, which areheavy loads for pharmaceutical companies.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a culture plate.

FIG. 2 illustrates a flow of sample preparation.

FIG. 3 illustrates fractions quantified in steps 4, 5, and 6 and imageview of drug distribution.

FIG. 4 illustrates definitions of fractions and results of CDF.

FIG. 5 illustrates the distribution ratio of CDF.

FIG. 6 illustrates the distribution ratio of Rhodamine 123.

FIG. 7 illustrates a configuration of an automatic measuring apparatus.

FIG. 8 is a top view of a sample preparation unit.

FIG. 9 is a front view of the sample preparation unit.

FIG. 10A is a flow chart of operations of the automatic measuringapparatus.

FIG. 10B is a flow chart of operations of the automatic measuringapparatus.

FIG. 11 illustrates chipheads and chips.

DESCRIPTION OF EMBODIMENTS Embodiment 1

In this embodiment, the aforementioned method of componential analysisfor evaluation of efficacy is described in connection with steps 0-6 inFIG. 2 . Steps 4-6 are described in detail referring to FIG. 3 .

In the following plural embodiments, the present invention is describedin connection with methods for evaluating medicinal properties. However,these are examples for illustrating the claimed invention and themethods of componential analysis according to the claimed invention canbe, needless to say, used to evaluate chemical substances other thandrug and also to evaluate drug metabolites. Moreover, specificconditions such as buffer solution, temperature, and time indicated inthese embodiments are for the illustration purpose and other conditionshaving similar effects without departing from the technical idea of thepresent invention can be, needless to say, used.

This embodiment describes a method using at least 3 sequences includingsequences 1-3. Each sequence contains a holding region for holdingcells. These holding regions may contain separate containers forrespective sequences or one container may be divided into a plurality ofholding regions for respective sequences.

<Step 0: Preparation and Culture of Hepatocytes>

In this step, preparation and culture of hepatocytes are conducted toexamine the effect of the drug. An example is illustrated below.

Hepatocytes were prepared by in situ collagenase perfusion method. Thedetails are as follows. A rat (5-6 week-old) is anesthetized withpentobarbital and laparotomy is performed to insert a catheter into theportal vein and inject the preperfusion solution (a Ca²⁺ and Mg²⁺ freeHanks' solution containing EGTA). After confirming that blood issufficiently removed from the liver, the perfusion is stopped. Theperfusate is changed into a collagenase solution to conduct perfusion.In this embodiment, the perfusion is conducted with a Hanks' solutioncontaining 0.05% collagenase, but the collagenase solution is notlimited to this. Once confirmed that the tissue between the cells wasdigested by collagenase, the perfusion is stopped. The liver is excised,sliced in a cooled Hanks' solution, and separated into cells bypipetting. Damaged hepatocytes are removed by centrifugation at 500 Gfor 5 minutes with an isotonic Percoll solution. The viability of theresultant hepatocytes is determined by the trypan blue-exclusion test.Hepatocytes having a viability of 85% or more are used for culture.Here, hepatocytes having a viability of 85% or more are used forculture, but such culture is, needless to say, not necessarily limitedto the condition. In addition, the preparation of hepatocytes is notnecessarily limited to that by in situ collagenase perfusion method. Thehepatocytes to be used are not limited to those derived from a rat andthe strain of rat is not limited. This embodiment uses hepatocytes, butcells are not limited to this.

Hepatocytes prepared by in situ collagenase perfusion method asdescribed above are suspended in a medium. The hepatocytes are suspendedat a density of 5×10⁵ cells/mL in a medium and plated onto acommercially available culture dish coated with collagen. The densityupon plating, the medium, and the culture plate 001 are not particularlylimited. The culture plate is illustrated in FIG. 1 . A 24 well cultureplate having 24 culture regions (wells, 002) is here illustrated, butthe culture plate is not limited to this as long as it can containpredetermined cells and containers in other shapes may be used. Afterplating, culture is started under conditions of 5% CO₂ and 37° C. usinga CO₂ incubator. After 18 hours or more, the first medium change isconducted. Although the medium to be used for culture 18 hours after theplating is not particularly limited, in this embodiment, the medium(FCS−), a medium in which FCS is removed from the medium (10% FCS+),supplemented with Matrigel is used. After this, medium change with themedium (FCS−) is conducted every 24 hours. As described later, testingis conducted under three different conditions (sequences 1, 2, and 3:described in detail in step 5) in step 5, three independent cultureplates with the same conditions are prepared at this point. In steps 0to 4 and step 5, the same testing operations are conducted in all thethree sequences.

<Step 1: Conditioning of Hepatocytes>

In this step, the cells cultured in step 0 are conditioned in a suitablecondition for the drug evaluation. An example is illustrated below.

The culture supernatant of the cells cultured in step 0 for 4 days isremoved and 400 μL of Hanks' solution is added as a buffer. The cellsare incubated at 37° C. for 10 minutes (FIG. 2 , step 1). The type andamount of buffer are not particularly limited.

Preferably, the operations of step 1 are repeated twice. By replacingthe medium used in the culture of step 0 with buffer solution (e.g.,Hanks' solution) and adapting the cells to the buffer ingredients, theyare conditioned for the accurate measurement and analysis in thefollowing steps. The number of times step 1 is repeated can be, needlessto say, changed depending on the types of buffer solution and cellsused.

<Step 2: Administration of Drug Solution>

In this step, the drug solution to be evaluated is administered tocells. An example is illustrated below.

The buffer is removed, and then 200 μL of 10 μM CDF (a fluorescentreagent) is added to the well. The plate is incubated at 37° C. for 30minutes and then maintained at 4° C. for 5 minutes (FIG. 2 , step 2).The type, concentration, and amount of the reagent are not particularlylimited. CDF emits fluorescence and can be therefore easily quantifiedby a plate reader as a model reagent. The amount of administration, 200μL, was chosen so that the cells as a whole in the well would beimmersed in the reagent. The concentration of CDF may be a concentrationconventionally used for a fluorescence assay of cells. Thisconcentration is preferably applied as an amount of the reagent for thedetection with a plate reader. 30 minutes of the incubation time wasdetermined based on the result of a preliminary examination that thetime required for getting the equilibrium between uptake and excretionof a drug is 30 minutes. The plate temperature was lowered to 4° C.after incubation for 30 minutes for the purpose of preventing theleakage of the administered drug from the cells. This is becauselowering the temperature in the container prevents cells from excretingthe drug.

As a result of thus lowering the temperature of drug to a predeterminedtemperature or lower, the drug remains in the cells and the leakage ofthe drug from the cells can be suppressed. In this embodiment, thetemperature is lowered to 4° C., but the temperature may be anytemperature as long as the leakage of the administered drug from thecells can be suppressed at the temperature. The method for thesuppression is not limited to lowering the temperature, but can be anymethod by which leakage of the administered drug from the cells can besuppressed, for example, by administering an inhibitor. In any case, thetemperature in step 2 is lower than the plate temperature in step 1.This is because the temperature for the conditioning in step 1 ispreferably a temperature that activates absorption and excretion of thedrug by cells (e.g., 37° C.), in contrast to step 2.

The timing of the drug administration is not limited to that of thisembodiment. For example, the drug may be administered on the day beforeor a few days before the testing day. In this case, the drug may beadministered in step 0 and steps 1 and 2 can be omitted.

<Step 3: Washing of Cells>

In this step, the drug solution other than that transferred into thecells in step 3 is washed away. An example is described below.

The cells were then washed 3 times with 400 μL, of ice-cold Hanks'solution while keeping the plate at 4° C. (FIG. 2 , step 3). The purposeof keeping the plate at 4° C. is the same as that described above. Theamount of the Hanks' solution for washing was determined to be 400 μL,for the purpose of removing medium ingredients remaining on the innerwall of the well because the amount of the medium in culture is 400 μL.The number of washing in general biochemistry assays is usually threetimes, which is adopted here. Conditions for the washing are notlimited. This step removes anything other than the drug solution to bemeasured, making it possible to improve the accuracy of the efficacyevaluation in the later steps.

<Step 4: Collection of Fraction of Basolateral Efflux>

As an index for evaluating efficacy, it is effective to analyze whetherthe administered drug tends to remain in the cell for a long time oronly for a short time. Therefore, this step aims to obtain the data forthe aforementioned index and the drug is leaked from the cells in apredetermined time before evaluating the cells in respective sequencesin step 5 described later. An example is illustrated below.

A buffer solution (e.g., Hanks' solution) for the pretreatment is firstadministered, the plate is incubated at 37° C. for 30 minutes, and theHanks' solution (supernatant) containing the drug was collected (FIG. 2, step 4). The buffer solution may be the same as or different from thebuffer solution used in step 5. The plate was maintained at 37° C. sothat the drug accumulated in the hepatocytes (FIG. 3, 101 ) by step 3 isexcreted into the Hanks' solution via the first basolateral efflux, thatis, the passively diffusion (FIG. 2 , step 4, (1)′) and/or via thetransporter (TP) (FIG. 2 , step 4, (2)′ and 102 in the image view).Because basolateral efflux occurs also in step 5 as described later, forthe distinguishment, the basolateral efflux in step 4 is defined as thefirst basolateral efflux and the basolateral efflux in step 5 as thesecond basolateral efflux. Essentially, both steps represent thebasolateral efflux(supernatant). Transporters are membrane proteins thatare responsible for transportation of substance and expressed on thecell membrane. They are responsible for active transportation ofsubstance between inside and outside of the cell. The passive diffusionis excretion to outside of the cell other than that via the transporterand includes the leakage through the cell membrane. The drug excretedinto the Hanks' solution in step 4 was defined as a fraction ofbasolateral efflux, because the upper surface of the cells facing theHanks' solution corresponds to the basal/basolateral face (FIG. 3, 104), which is considered to be a part facing the blood vessel in the body.

Step 5, which is explained next, includes collecting the drug in threedifferent operations (sequences 1, 2, and 3) and an index for examiningwhether the drug tends to remain in the cell can be acquired by, priorto step 5, obtaining the first fraction of basolateral efflux (the drugexcreted into Hanks' solution) in step 4 as mentioned above.

The Hanks' solution used to obtain the fraction of the basolateralefflux may be of any sequence. For example, Hanks' solutions of allsequences 1-3 described later or a part thereof may be evaluated.

Steps 3 and 4 are steps of conducting the analysis according to thepresent invention in more detail to conduct highly accurate evaluation.The present invention can be carried out skipping steps 3 and 4 andconducting operations of step 5. The aforementioned steps 0-4 areconducted in at least sequences 1-3, as shown in FIG. 2 . In step 5,operations are conducted under different conditions for sequences 1-3 soas to obtain a plurality of index data sought in the present inventionby the analysis described later. An example is illustrated below.

<Step 5, Sequence 1: Collection of Supernatant in 37° C. DisruptedSystem>

In sequence 1, as shown in sequence 1 in FIG. 3 , the drug is excretedfrom the cell into a buffer solution (e.g., Hanks' (-) solution) in anamount equal to the sum of biliary excretion (3) and the secondbasolateral efflux (that is, diffusion (1) and excretion via transporter(TP) (2)) discharge. Hanks' (-) solution is Hanks' solution free ofcalcium and magnesium ions and used, for example, when cell-celladhesion is not enhanced intentionally as in this sequence. For thepurpose of positively removing cell-cell adhesion, a chelator such asEGTA is used as described in the following. Next, 200 μL of Hanks' (-)solution containing 1 mM EGTA was added. EGTA has a chelating effect ofsuppressing the effect of Ca²⁺ and Mg²⁺ involved in the adhesion ofintercellular adhesion molecules and is a reagent for removing cell-celladhesion. This causes the disruption of bile ducts formed during theculture process. The area was incubated at 37° C. for 30 minutes, andthen the supernatant was collected. The chelating reagent is not limitedto EGTA, as long as it has a chelating effect. Type and amount of thebuffer containing EGTA and temperature and time of the incubation arenot particularly limited. In sequence 1 of step 5, the drug remaining inthe cells (FIG. 3 , step 5 and the image drawing, (1) and (2)) and thedrug excreted into the bile canaliculi (FIG. 3 , step 5 and the imagedrawing, (3)) upon the completion of step 4 are all excreted into thesupernatant and collected (FIG. 3 , step 5, sequence 1). In step 5, (3)is defined as a biliary-excreted fraction, because the cell membranepart around the gap formed in the cell-cell adhesion part corresponds toan apical face (FIG. 3, 105 ) and the gap is assumed to be a bilecanaliculus (FIG. 3, 103 ). In step 5, sequence 1, the temperature ismaintained at 37° C., and therefore the fractions excreted from thecells to the basolateral side include the fraction via passive diffusion(FIG. 3 , step 5, (1)) and the fraction via the transporter (FIG. 3 ,step 5, (2)).

<Step 5, Sequence 2: Collection of Supernatant in 37° C. MaintainedSystem>

In sequence 2, bile ducts are not disrupted as in sequence 1 and thedrug is excreted only by the second basolateral efflux (diffusion (1)and transporter (2)) indicated in FIG. 3 .

200 μL of Hanks' solution was added. In sequence 2, any chelating agentsuch as EGTA is not included as in sequence 1, and therefore cell-celladhesion is maintained. Accordingly, sequence 2 is a sequence where thedisruption of bile canaliculi is not induced. The area was incubated at37° C. for 30 minutes, and then the supernatant was collected. Insequence 2 in step 5, the drug remaining in the cells (FIG. 3 , step 5and the image view, (1) and (2)) upon the completion of step 4 isexcreted into the supernatant and collected (FIG. 3 , step 5, sequence2). Following the foregoing description, the drug excreted into thesupernatant is defined as a second fraction of basolateral efflux. Instep 5, sequence 2, the temperature is maintained at 37° C., andtherefore the fractions excreted from the cells to the basolateral sideinclude the fraction via passive diffusion (FIG. 3 , step 5, (1)) andthe fraction via the transporter (FIG. 3 , step 5, (2)).

By quantitatively analyzing the amounts of the drug collected fromsequence 1 and sequence 2 in step 5, for example, by subtracting thequantified value of the amount of the drug collected in sequence 2 fromthe quantified value of the amount of the drug collected in sequence 1,each of the amount of the drug excreted to the bile canaliculi, (3) andthe amount of the drug of the second fraction of basolateral efflux canbe calculated.

<Step 5, Sequence 3: Collection of Supernatant in 4° C. MaintainedSystem>

Sequence 3 has a temperature condition lower than that in sequences 1and 2 as described later, and thereby the excretion of the drug via thetransporter (1) is suppressed and only the amount of the drug excretedby diffusion (2) is excreted in the second fraction of basolateralefflux.

200 μL of Hanks' solution was added. The area was incubated at 4° C. for30 minutes, and then the supernatant was collected. The testingtemperature is different from step 5 sequence 2, which is also amaintained system. This aims to suppress basolateral efflux via thetransporter by cooling to 4° C. In step 5, sequence 3, a sequence havinga low temperature of 4° C. is adopted to suppress the transporteractivity, and thereby quantify only the amount excreted by passivediffusion (FIG. 3 , step 5, (1)). Comparing this with step 5, sequence 2makes it possible to calculate (quantify) the amount of the basolateralefflux fraction via transporter. For each drug, a specific transporteris present for moving between inside and outside of the cell. Therefore,the ability to exclude the amount of excretion by passive diffusion andquantify the amount of excretion via the transporter means the abilityto quantify the amount of excretion specific for the drug. Type andamount of the buffer and temperature and time of the incubation are notparticularly limited. As described above, the fraction excreted via thetransporter can be calculated by quantifying the drug collected fromsequence 2 and sequence 3 in step 5. Although the calculation ofexcreted fractions using sequences 1, 2, and 3 is illustrated in thisembodiment, the number of sequences is not limited to three. Forexample, when only the separation of biliary excretion (3) and thesecond basolateral efflux ((1)+(2)) is desired, it is sufficient toconduct only sequences 1 and 2; and when only the separation of thediffusion (1) and the transporter (2) in the second basolateral effluxis desired, it is sufficient to conduct only sequences 2 and 3. It isalso possible to measure only the amount excreted by diffusion byadditionally having a disrupted system at 4° C., as needed, to suppressbiliary excretion in excretion via transporter. For example, comparisonof this 4° C. disrupted system with sequence 3 makes it possible toseparately evaluate the biliary excretion by diffusion and the biliaryexcretion via transporter.

The steps in each of sequences 1-3 may be conducted in any order andsteps may be conducted in parallel.

<Step 6: Collection of Biliary Fraction and Intracellular Fraction>

In step 6, the same operations are conducted for the 3 sequences again.In step 6, the drug collection for quantifying the fraction remaining inthe cells is conducted. When fluorescent agent is quantified using aplate reader, it is preferred that, for example, 200 μL of a Hankssolution containing 1% surfactants is added and suspended and the totalamount is collected (FIG. 2 , step 6). This disrupts the cell membraneand makes it possible to have the drug remaining in the cells excreted.The resultant samples are transferred to a culture plate andfluorescence was measured using a plate reader. Wells containing onlythe Hanks' solution are prepared for the blank measurement. Fluorescenceintensity is measured with an excitation wavelength of 484 nm and anabsorption wavelength of 519 nm.

When the drug is quantified using a mass spectrometry (LCMS) apparatus,water is added for lowering the tonicity, the fraction remaining in thecells are extracted by a treatment with an organic solvent and thensuspended with an organic solvent such as methanol, and the total amountis collected (FIG. 2 , step 6). Quantification of the drug issubsequently conducted using an LCMS apparatus as described later.

<Determination of Distribution Ratio and Scoring of Respective FractionsBased on Result of Measurement>

The distribution ratio to respective fractions is calculated based onthe fluorescence intensities reflecting the amounts of the drug obtainedin the aforementioned steps. Here, the sum of steps 5 and 6 (FIG. 3 , B#+C #=(1)+(2)+(3)+(4)) is defined as 100% amount of the drug. This isreferred to as “pattern 1.”

From the six values (B1, B2, B3, C1, C2, C3) obtained by the measurementof fluorescent amount, the distribution ratio of fractions illustratedin pattern 1 in FIG. 4 is obtained. As for these values,

the second fraction of the basolateral efflux only by diffusion(Extracellular Efflux by Diffusion, ExEfx-Dif) is expressed as B3;

the second fraction of the basolateral efflux only by the transporter(Extracellular Efflux by Transporter, ExEfx-TP) is expressed as B2-B3;

the fraction of the biliary excretion (Bile Canaliculi Efflux, BCEfx) isexpressed as B1-B2; and

the fraction remaining in the cells (Cell) is expressed as C1.

Based on this result, a circle graph such as pattern 1 in FIG. 5 can begenerated and visual understanding of a total picture of thedistribution ratio of respective fractions becomes possible.

Furthermore, scoring of particular drugs as the following becomespossible based on the quantification result. In this embodiment, thescoring calculated for CDF is illustrated. The calculated scores are notlimited to these.

A score for evaluating basolateral efflux via transporter is calculatedas

ratio of the amount of the drug excreted via transporter to the amountof the drug of the second fraction of basolateral efflux (Ratio ofExtracellular Efflux by Diffusion, RexEMTP), (B2-B3)/B2.

A score for evaluating the biliary excretion can be calculated as

ratio of the amount of the drug excreted into the bile canaliculi to thetotal amount of the drug taken up into the cells (Biliary RetentionDrug, BiRD), (B1−B2)/(C1+C2), or alternatively,

ratio of the amount of the drug excreted into bile canaliculi to theamount of the drug remaining in the cells, (C2−C1)/C2 or (B1−B2)/C1, orthe like.

<Comparison of Distribution Ratios and Scores Between Different Drugs>

The operations same as those described above can be conducted usingRhodamine123 instead of CDF to obtain the result shown in FIG. 6(pattern 1). The result indicates that a distribution ratio apparentlydifferent from the result of CDF shown in FIG. 5 (pattern 1) can bedetected.

For example, the ratio of the amount of drug excreted via transporter tothe amount of drug excreted to the basolateral side (RexEMTP) and theratio of the amount of drug excreted into the bile duct to the totalamount of drug taken up into the cells (BiRD) for CDF are 41.08 and18.58, respectively, while those for Rhodamine123 are 52.52 and 4.92,respectively, indicating that CDF is a drug that has an strongertendency to be excreted into the bile canaliculi but not to thebasolateral side than Rhodamine123 does. As described above, the presentinvention makes it possible to evaluate compounds on thepharmacokinetics they exhibit.

Embodiment 2

Embodiment 2 describes the determination of distribution ratio andscoring of respective fractions based on the measurement result when amethod different from embodiment 1 is used.

In this embodiment, a quantified value S # (corresponding to step 4)described in FIG. 3 , in addition to the distribution ratio ofrespective fractions shown in pattern 1 in embodiment 1, is used toobtain information on whether the administered drug has a strong or weaktendency to remain in the cells, which makes more accurate evaluationpossible. Accordingly, the sum of steps 4, 5, and 6 (FIG. 3 , S #+B #+C#=(1)′+(2)′+(1)+(2)+(3)+(4)) is defined as the 100% amount of theadministration drug. This is referred to as “pattern 2”. Accordingly,nine values (S1, S2, S3, B1, B2, B3, C1, C2, C3) obtained in thesequences in steps 4, 5, and 6 can be used as evaluation indexes.

From nine values obtained from measurement of fluorescent amounts, thedistribution ratio of respective fractions shown in pattern 2 in FIG. 4is obtained. As for these values,

the first fraction of the basolateral efflux (Sup fraction) is expressedas S1 (≈S2≈S3);

the second fraction of the basolateral efflux only by diffusion(Extracellular Efflux by Diffusion, ExEfx-Dif) is expressed as B3;

the second fraction of the basolateral efflux only by the transporter(Extracellular Efflux by Transporter, ExEfx-TP) is expressed as B2-B3;

the fraction of the biliary excretion (Bile Canaliculi Efflux, BCEfx) isexpressed as B1-B2; and

the fraction remaining in the cells (Cell) is expressed as C1.

Based on this result, a circle graph such as pattern 2 in FIG. 5 can begenerated and visual understanding of a total picture of thedistribution ratio of respective fractions becomes possible. Asdetermined by a method described in embodiment 2, the Sup fraction,which is an index for tendency to remain or not to remain in the cells,for CDF is 70.82, while that for Rhodamine123 is 39.29 (FIG. 6 , pattern2), indicating that CDF has a tendency to be excreted to the basolateralside. This is consistent with the evaluation of the fraction remainingin the cells in embodiment 1.

Furthermore, scoring of characteristics of drugs becomes possible basedon the quantification result, as described in embodiment 1.

Embodiment 3

Embodiment 3 describes an example of apparatus automating a series ofsteps described in embodiments 1 and 2. About purposes of operations andfunctions of configurations of the apparatus described later,descriptions may be omitted when the descriptions are overlapped withthose of the aforementioned embodiments 1 and 2.

In the configurations of the apparatus described later, the regionsholding cells referred to as sequences 1-3 shown in embodiments 1-2 aredescribed using expressions such as “a well for sequence 1,” “a well forsequence 2,” and “a well for sequence 3.” For example, a plurality ofcontainers in the aforementioned “well culture plate” may be defined forrespective sequences or a plurality of wells in a well culture plate maybe divided and the divided regions may be defined as, for example, “afirst container,” “a second container,” and “a third container.”

As for the well for sequence 1 and the well for sequence 2 inparticular, effective evaluation may be provided in terms of accuracyand speed by defining them as wells in divisions of one well cultureplate because they are subjected to a substantially the same temperaturecontrol.

As for the operations of the apparatus described latter, exchange ofwells (containers) for respective sequences may be automatic or manual.Descriptions on exchange and placement of the containers will be omittedin the following description.

The apparatus includes a culture unit 106, a sample preparation unit 107(including an input unit 107A), an analyzing unit 108 and a display unit109, as shown in FIG. 7 . The sample preparation unit 107 has atemperature controlling unit 107B for controlling temperature inrespective containers (plates) described later, a liquid-feeding unit107C capable of supplying or collecting liquid in a container, and thelike. The analyzing unit 108 has a measurement unit 108A for measuringthe amount of a component such as a drug and analysis unit 108B foranalyzing each of: an amount excreted via transporter, an amountexcreted via the bile duct, an amount remaining in the cells, and anamount excreted via a route (diffusion) other than the transporter andthe bile duct based on the amount of the component such as a drugobtained in the measurement unit. The above conformation of theapparatus is an example and, for example, other configurations may be,needless to say, adopted, for example, one in which only the analysisunit operates in a separate apparatus and the information obtained bythe measurement unit is transmitted to the separate apparatus.

A detailed configuration of the sample preparation unit is shown inFIGS. 8 and 9 . The sample preparation unit aims to prepare respectivefractions to be analyzed automatically as described in embodiments 1-2.Respective components will be described in connection with the flowchart described later.

FIG. 10 is a flow chart of operations of the automatic measuringapparatus. The flow chart of FIG. 10 is merely an example and theoperations may be different from this when a timing of the drugadministration is different as described in embodiment 1, <step 2>. Inthis embodiment, plates having a plurality of cell holding regions(wells) are used as an example for illustrating the containers forholding cells, but the containers are not limited to plates and anycontainers that can contain cells may be, needless to say, used.

<Transfer from Culture Unit to Sample Preparation Unit>

Hepatocytes are first cultured in the culture unit 106 (FIGS. 7 and 10 )(FIG. 10(a) substep 1). Subsequently, the plates containing the culturedhepatocytes are transferred on a plate holder 210 having a firsttemperature controlling function and a plate holder (211) having asecond temperature controlling function in the sample preparation unit(FIGS. 7 and 10 ) (FIG. 10(a), substep 2).

<Sample Preparation: Corresponding to Step 1 in Embodiments 1 and 2>

An aspiration head 205, equipped with an aspiration nozzle to aspirateliquid, installed in the liquid-feeding unit 107C moves to a well 002,filled with medium to be removed, in a culture plate 001 on theaforementioned plate holder and aspirates the medium from the well toremove the total amount (FIG. 10 , substep 3). The removed medium iscollected (discarded) in the waste liquid tank 206.

Next, a tip 302 for containing liquid 301 from a tip rack 207 storing aplurality of tips is attached onto a tiphead 204 installed to anaspiration nozzle. The tiphead moves to a room temperature drug solutionrack 209 and aspirate a buffer (FIG. 10 , substep 4). The tiphead movesto a target well and introduces the buffer (FIG. 10(a), substep 5) andthen the tiphead moves to a dust box 214 and discards the tip. Anexchangeable tip is used here to prevent contaminations, but the tip isnot limited to this.

The absorption head moves to the well filled with the buffer and removesthe buffer (FIG. 10(a), substep 6). The removed medium is collected anddiscarded in the waste liquid tank 206. This step is repeated twice intotal (washing step) (FIG. 10(a), substep 7).

Next, a tip in the tip rack 207 is attached onto the tiphead 204, thetiphead moves to the room temperature drug solution rack 209, andaspirates the buffer (FIG. 9 , substep 8). The tiphead moves to thetarget well and introduces the buffer and then the tiphead moves to thedust box 214 and discards the tip. The plate is allowed to stand at 37°C. for 10 minutes (conditioning) (FIG. 10(a), substep 9). Subsequently,the aspiration head 205 moves to the target well filled with the bufferand removes the total amount of the buffer (FIG. 9 (a), sub step 10).

<Sample Preparation: Corresponding to Step 2 in Embodiments 1 and 2>

A tip in the tip rack 207 is attached onto the tiphead 204, the tipheadmoves to the room temperature drug solution rack 209, and aspirates thedrug solution (FIG. 10(a), substep 11). The tiphead moves to the targetwell and introduces the drug solution (FIG. 10(a), substep 12) and thenthe tiphead 204 moves to the dust box 214 and discards the tip. Theplate is allowed to stand at 37° C. for 30 minutes (FIG. 10(a), substep12). Subsequently, the temperature of the plate holder 210 having thefirst temperature controlling function and the plate holder 211 havingthe second temperature controlling function, which are an exampleconfiguration of the container holding unit for holding containers, ischanged from 37° C. to 4° C. (FIG. 10(a), substep 13), and then theaspiration head 205 moves to the well filled with the drug solution andremoves a total amount of the drug solution (FIG. 10(a), substep 14).Although the temperature controlling unit in this embodiment isdescribed as a plate folder having a temperature controlling function,the temperature controlling unit may be, needless to say, separated fromthe container holding unit.

<Sample Preparation: Corresponding to Step 3 in Embodiments 1 and 2>

A tip in the tip rack 207 is attached onto the tiphead 204, the tipheadmoves to a refrigerated drug solution rack 208, and aspirates a buffer(FIG. 10(a), substep 15). A refrigerated drug solution is used to stopactive biological phenomena such as the transporter activity, asdescribed above. The refrigerated drug solution rack 208 aims to hold adrug solution, a buffer, and the like at a low temperature for thepurpose. The tiphead moves to the target well and introduces the buffer(FIG. 10(a), substep 16) and then the tiphead 204 moves to the dust box214 and discards the tip. The absorption head moves to the well filledwith the buffer and removes the buffer (FIG. 10(a), substep 17). Theremoved medium is discarded in the waste liquid tank 206. This step isrepeated three times in total (washing step) (FIG. 10(a), substep 18).

<Sample Preparation: Corresponding to Step 4 in Embodiments 1 and 2>

The temperature of the plate holder 210 having the first temperaturecontrolling function and the plate holder 211 having the secondtemperature controlling function is changed from 4° C. to 37° C. (FIG.10(a), substep 19), a tip in the tip rack 207 is attached onto thetiphead 204, and the tiphead moves to a refrigerated drug solution rack208 and aspirate the buffer (FIG. 10(a), substep 20). The tiphead movesto the target well and introduces the buffer (FIG. 10(a), substep 21)and then the tiphead 204 moves to the dust box 214 and discards the tip.The plate is allowed to stand at 37° C. for 30 minutes (FIG. 10(a),substep 20). Subsequently, the temperature of the plate holder 210having the first temperature controlling function and the plate holder211 having the second temperature controlling function is changed from37° C. to 4° C. (FIG. 10(a), substep 22), and then a tip in the tip rack207 is attached onto the tiphead 204, the tiphead moves to the wellfilled with the buffer containing the drug, aspirates the buffer(supernatant) containing the drug, and dispenses it into collectionplates for collection on the first plate holder 212 and the second plateholder 213 (collection) (FIG. 10(a), substep 23).

<Sample Preparation: Corresponding to Step 5 in Embodiments 1 and 2>

The temperature of the plate holder 210 having the first temperaturecontrolling function is changed from 4° C. to 37° C. (FIG. 10(a),substep 24), a tip in the tip rack 207 is attached onto the tiphead 204,and the tiphead moves to the room temperature drug solution rack (209)and aspirates a buffer containing EGTA (FIG. 10(a), substep 24). The tiphead moves to a well for sequence 1 on the first plate holder 212, thebuffer containing EGTA is introduced (FIG. 10(b), substep 25), then thetiphead 204 moves to the dust box 214, and the tip is discarded. Theplate is allowed to stand at 37° C. for 30 minutes (FIG. 10(b), substep26).

A tip in the tip rack 207 is attached onto the tiphead 204, the tipheadmoves to the room temperature drug solution rack 209, and aspirates thebuffer (FIG. 10(b), substep 27). The tip head moves to a well forsequence 2 on the first plate holder 212, the buffer is introduced (FIG.10(b), substep 28), then the tiphead 204 moves to the dust box 214, andthe tip is discarded. The plate is allowed to stand at 37° C. for 30minutes (FIG. 10(b), substep 28).

A tip in the tip rack 207 is attached onto the tiphead 204, the tipheadmoves to the refrigerated drug solution rack 209, and aspirates thebuffer (FIG. 10(b), substep 29).

The tip head moves to a well for sequence 3 on the second plate holder213, the buffer is introduced (FIG. 10(b), substep 30), then the tiphead204 moves to the dust box 214, and the tip is discarded. The plate isallowed to stand at 4° C. for 30 minutes (FIG. 10(b), sub step 30).

A tip in the tip rack 207 is attached onto the tiphead 204 and thetiphead moves to the well filled with an EGTA buffer containing thedrug, aspirates the EGTA buffer (supernatant) containing the drug, anddispenses (collects) the buffer into a collection plate for collectionon the first plate holder 212 (FIG. 10(b), substep 31).

A tip in the tip rack 207 is attached onto the tiphead 204 and thetiphead moves to the well filled with the buffer containing the drug,aspirates the buffer (supernatant) containing the drug, and dispenses(collects) the buffer into a collection plate for collection on thefirst plate holder 212 (FIG. 10(b), substep 32).

A tip in the tip rack 207 is attached onto the tiphead 204, the tipheadmoves to the well filled with the EGTA buffer containing the drug,aspirates the buffer (supernatant) containing the drug, and dispenses(collects) the buffer into the collection plate for collection on thefirst plate holder 213 (FIG. 10(b), substep 33).

The temperatures of the plate holder 210 having the first temperaturecontrolling function and the plate holder 211 having the secondtemperature controlling function are both changed to room temperature(FIG. 10(b), substep 34), a tip in the tip rack 207 is attached onto thetiphead 204, and the tiphead moves to the room temperature drug solutionrack 209 and aspirates 1% TritonX-100 or pure water/methanol (FIG.10(b), substep 35). The tip head moves to wells for sequences 1, 2, and3 on the plate holder (210) having the first temperature controllingfunction and the plate holder 211 having the second temperaturecontrolling function, and introduces 1% TritonX-100 or purewater/methanol (FIG. 10(b), substep 36), the tiphead 204 moves to thedust box 214, and the tip is discarded.

A tip in the tip rack 207 is attached onto the tiphead 204 and thetiphead moves to a well filled with the aforementioned reagent,aspirates the total amount of the cell suspension, and dispenses thesuspension into the collection plates for collection on the first plateholder 212 and the second plate holder 213 (collection) (FIG. 10(b),substep 37).

The operations of the aforementioned apparatus have been described asthe respective operations of sequences 1-3 in embodiments 1 and 2 areconducted in order. The steps may be conducted in sequence or the stepsmay be conducted in parallel.

<Transfer from Sample Preparation Unit to Measurement Unit>

The collected drug in the culture plate is transferred to themeasurement unit (FIG. 10(b), substep 38). In the measurement unit, themeasurement of the drug by a plate reader or LCMS is conducted (FIG.10(b), sub step 39).

<Calculation of Distribution Ratio and Score and Display of Result byAnalysis Unit>

The distribution ratio and the score of respective fractions arecalculated based on the measurement results (FIG. 10(b), substep 40).Then, the resultant calculated values are displayed on a display unit(FIG. 10(b), sub step 41).

Examples of the foregoing configurations described in connection withembodiments 1-3 include, for example:

<Configuration 1>

A componential analyzer, comprising: a holding unit for holding aplurality of containers holding a predetermined cell; a temperaturecontrolling unit for controlling temperatures in the plurality ofcontainers; and an analyzing unit for measuring a component in theplurality of containers and analyzing the measured component, whereinthe plurality of containers is at least a first container and a secondcontainer; the first container and the second container each contain afirst buffer solution; the temperature controlling unit controlstemperature in the first container and temperature in the secondcontainer so that the temperatures become different from each other; andwherein the analyzing unit measures: an amount of the component excretedfrom a cell in the first container to the first buffer solution in thefirst container and an amount of the component excreted from a cell inthe second container to the first buffer solution in the secondcontainer, and the analyzing unit analyzes an amount of the componentexcreted via transporters in the cells.

<Configuration 2>

The componential analyzer according to Configuration 1, wherein thepredetermined cell is s hepatocyte; the plurality of containerscomprises at least a first container, a second container, and a thirdcontainer; the third container contains a second buffer solutionsupplemented with a predetermined substance;

the temperature controlling unit controls a first temperature in thefirst container and in the third container and a second temperature inthe second container so that the second temperature is lower than thefirst temperature; wherein the analyzing unit measures: an amount of thecomponent excreted from the hepatocyte in the first container to thefirst buffer solution in the first container, an amount of the componentexcreted from the hepatocyte in the second container to the first buffersolution in the second container, and an amount of the componentexcreted from the hepatocyte in the third container to the second buffersolution in the third container, and the analyzing unit analyzes: anamount of the component excreted via the transporters in the hepatocyte,an amount of the component excreted via the bile canaliculus of thehepatocyte, and an amount of the component excreted via a route otherthan the transporter and the bile canaliculus of the hepatocyte.

<Configuration 2>

The componential analyzer according to Configuration 2, furthercomprising a liquid-feeding unit for supplying or collecting a liquid inthe plurality of containers, wherein the liquid-feeding unit: suppliesthe plurality of containers with a component solution comprising thecomponent, collects the component solution and then supplies theplurality of containers with a third buffer solution, and collects thethird buffer solution and then supplies the first container and thesecond container with the first buffer solution and the third containerwith the second buffer solution; and wherein the analyzing unit measuresan amount of the component excreted into the third buffer solution fromthe hepatocyte contained in either of the plurality of containers.

<Configuration 4>

The componential analyzer according to Configuration 3, wherein thethird buffer solution and the first buffer solution are the same buffersolution.

<Configuration 5>

The componential analyzer according to Configuration 3, wherein thetemperature controlling unit controls temperature in at least onecontainer of the plurality of containers when the at least one containercontains the third buffer solution to be lower than the firsttemperature.

<Configuration 6>

The componential analyzer according to Configuration 1, wherein theanalyzing unit measures: an amount of the component remaining in thehepatocyte in the third container and an amount of the componentremaining in the hepatocyte in at least one container of the firstcontainer and the second container, and the analyzing unit analyzes anamount of the component remaining in other than bile canaliculi of thehepatocyte.

<Configuration 7>

A drug component analyzer, comprising: a holding unit for holding aplurality of containers containing a hepatocyte having absorbed a drug;a liquid-feeding unit for supplying a liquid in the plurality ofcontainers; a temperature controlling unit for controlling temperaturesin the plurality of containers; and an analyzing unit for measuring anamount of the drug in the plurality of containers and analyzing themeasured drug, wherein the plurality of containers comprises a firstcontainer, a second container, and a third container; wherein theliquid-feeding unit supplies the first container and the secondcontainer with a first buffer solution and the third container with asecond buffer solution promoting excretion of the drug from a bilecanaliculus of the hepatocyte; the temperature controlling unit controlsa temperature in the first container and in the third container and atemperature in the second container so that the later temperature islower than the former temperature; wherein the analyzing unit analyzeseach of: an amount of the drug excreted into the first buffer solutionfrom the hepatocyte in the first container, an amount of the drugexcreted into the first buffer solution from the hepatocyte in thesecond container, an amount of the drug excreted into the second buffersolution from the hepatocyte in the third container, an amount of thedrug excreted via transporters in the hepatocyte, an amount of the drugexcreted via bile canaliculi of the hepatocyte, and an amount of thecomponent excreted via a route other than the transporter and the bilecanaliculus of the hepatocyte.

<Configuration 8>

A drug component analyzer, comprising: a holding unit for holding aplurality of containers containing a hepatocyte having absorbed a drug;a liquid-feeding unit for supplying and discharging a liquid in theplurality of containers; a temperature controlling unit for controllingtemperatures in the plurality of containers; and an analyzing unit formeasuring an amount of the drug in the plurality of containers andanalyzing the measured drug, wherein the liquid-feeding unit: suppliesthe drug to the plurality of containers, discharges the drug from theplurality of containers, supplies a pretreatment buffer solution,collects the pretreatment buffer solution from the plurality ofcontainers and then supplies the first container and the secondcontainer of the plurality of culture containers with a first buffersolution, and the third container with a second buffer solutionpromoting excretion of the drug from a bile canaliculus of thehepatocyte; the temperature controlling unit controls a firsttemperature in the first container and in the third container and asecond temperature in the second container so that the secondtemperature is lower than the first temperature;

wherein the analyzing unit analyzes each of: an amount of the drugexcreted into the pretreatment buffer solution from the hepatocytes inthe plurality of containers, an amount of the drug excreted into thefirst buffer solution from the hepatocyte in the first container, anamount of the drug excreted into the first buffer solution from thehepatocyte in the second container, an amount of the drug excreted intothe second buffer solution from the hepatocyte in the third container,an amount of the drug excreted from the hepatocyte during pretreatmentof the hepatocytes, an amount of the drug excreted via transporters inthe hepatocyte, an amount of the drug excreted via bile canaliculi ofthe hepatocyte, and an amount of the drug excreted via a route otherthan the transporter and the bile canaliculus of the hepatocyte.

<Configuration 9>

The drug component analyzer according to Configuration 8, wherein thepretreatment buffer solution and the first buffer solution are the samebuffer solution.

<Configuration 10>

The drug component analyzer according to Configuration 8, wherein thetemperature controlling unit controls temperature in at least onecontainer of the plurality of containers when the at least one containercontains the pretreatment buffer solution to be lower than the firsttemperature.

<Configuration 11>

The drug component analyzer according to any one of Configurations 7 to10, wherein the analyzing unit measures: an amount of the componentremaining in the hepatocyte in the third container and an amount of thecomponent remaining in the hepatocyte in at least one container of thefirst container and the second container, and the analyzing unitanalyzes an amount of the component remaining in other than bilecanaliculi of the hepatocyte.

<Configuration 12>

A method of componential analysis, comprising: a temperature controllingstep of controlling temperatures in a first container containing apredetermined cell and in a second container containing thepredetermined cell so that the temperature in the second container islower than the temperature in the first container; a measuring step ofmeasuring an amount of a component excreted from the cell in the firstcontainer to the first buffer solution in the first container and anamount of the component excreted from the cell in the second containerto the first buffer solution in the second container; and an analyzingstep of analyzing an amount of the component excreted via transportersin the cells based on the result measured in the measuring step.

<Configuration 13>

The method of componential analysis according to Configuration 12,wherein the cell is a hepatocyte; the third container containing thehepatocyte comprises a second buffer solution supplemented with apredetermined substance; the temperature controlling step comprisescontrolling a first temperature in the first container and in the thirdcontainer and a second temperature in the second container so that thesecond temperature is lower than the first temperature; the measuringstep comprises measuring the followings: an amount of the componentexcreted from the hepatocyte in the first container to the first buffersolution in the first container, an amount of the component excretedfrom the hepatocyte in the second container to the first buffer solutionin the second container, and an amount of the component excreted fromthe hepatocyte in the third container to the second buffer solution inthe third container; and the analyzing step comprises analyzing each ofthe followings: an amount of the component excreted via the transportersin the hepatocyte, an amount of the component excreted via the bilecanaliculus of the hepatocyte, and an amount of the component excretedvia a route other than the transporter and the bile canaliculus of thehepatocyte.

<Configuration 14>

The method of componential analysis according to Configuration 13,further comprising: prior to the temperature controlling step, aliquid-feeding step of supplying or collecting a liquid in a pluralityof containers comprising at least the first container, the secondcontainer, and the third container; wherein the liquid-feeding stepcomprises: supplying the plurality of containers with a componentsolution comprising the component, collecting the component solution andthen supplying the plurality of containers with a third buffer solution,and collecting the third buffer solution and then supplying the firstcontainer and the second container with the first buffer solution andthe third container with the second buffer solution; and the measuringstep comprises analyzing an amount of the component excreted into thethird buffer solution from the hepatocyte contained in at least one ofthe plurality of containers.

<Configuration 15>

The method of componential analysis according to Configuration 14,wherein the third buffer solution and the first buffer solution are thesame buffer solution.

<Configuration 16>

The method of componential analysis according to Configuration 14,further comprising: prior to the temperature controlling step, apretreatment temperature controlling step of controlling a temperaturein at least one container of the plurality of containers when the atleast one container contains the third buffer solution to be lower thanthe first temperature.

<Configuration 17>

A method of analyzing a drug component, comprising: a liquid-feedingstep of supplying a liquid to a plurality of containers containing ahepatocyte having absorbed a drug; a temperature controlling step ofcontrolling temperatures in the plurality of containers; a measuringstep of measuring an amount of the drug in the plurality of containers;and an analyzing step of analyzing the drug measured in the measuringstep, wherein the plurality of containers comprises a first container, asecond container, and a third container; the liquid-feeding stepcomprises supplying the first container and the second container with afirst buffer solution and the third container with a second buffersolution promoting excretion of the drug from a bile canaliculus of thehepatocyte; the temperature controlling step comprises controlling atemperature in the first container and in the third container and atemperature in the second container so that the later temperature islower than the former temperature; and the measuring step comprisesmeasuring the followings: an amount of the drug excreted into the firstbuffer solution from the hepatocyte in the first container, an amount ofthe drug excreted into the first buffer solution from the hepatocyte inthe second container, an amount of the drug excreted into the secondbuffer solution from the hepatocyte in the third container, an amount ofthe component remaining in the hepatocyte in the third container and anamount of the component remaining in the hepatocyte in at least onecontainer of the first container and the second container; and theanalyzing step comprises analyzing each of the followings: an amount ofthe drug excreted via transporters in the hepatocytes, an amount of thedrug excreted via bile canaliculi of the hepatocytes, an amount of thedrug excreted via a route other than the transporter and the bilecanaliculus of the hepatocyte, and an amount of the component remainingin other than the bile canaliculus of the hepatocyte.

<Configuration 18>

A method of analyzing a drug component, comprising: a liquid-feedingstep of supplying or discharging a liquid in a plurality of containerscontaining a hepatocyte having absorbed a drug; a temperaturecontrolling step of controlling temperatures in the plurality ofcontainers; a measuring step of measuring an amount of the drug in theplurality of containers; and an analyzing step of analyzing the drugmeasured in the measuring step, wherein the liquid-feeding stepcomprises: supplying the drug to the plurality of containers,discharging the drug from the plurality of containers and supplying apretreatment buffer solution, collecting the pretreatment buffersolution from the plurality of containers and then supplying the firstcontainer and the second container of the plurality of culturecontainers with a first buffer solution, and the third container with asecond buffer solution promoting excretion of the drug from a bilecanaliculus of the hepatocyte; the temperature controlling stepcomprises controlling a first temperature in the first container and inthe third container and a second temperature in the second container sothat the second temperature is lower than the first temperature; themeasuring step comprises measuring the followings: an amount of the drugexcreted into the pretreatment buffer solution from the hepatocytes inthe plurality of containers, an amount of the drug excreted into thefirst buffer solution from the hepatocyte in the first container, anamount of the drug excreted into the first buffer solution from thehepatocyte in the second container, an amount of the drug excreted intothe second buffer solution from the hepatocyte in the third container,an amount of the component remaining in the hepatocyte in the thirdcontainer and an amount of the component remaining in the hepatocyte inat least one container of the first container and the second container;and the analyzing step comprises analyzing each of the followings: anamount of the drug excreted from the hepatocytes during pretreatment ofthe hepatocytes, an amount of the drug excreted via transporters in thehepatocytes, an amount of the drug excreted via bile canaliculi of thehepatocytes, an amount of the drug excreted via a route other than thetransporter and the bile canaliculus of the hepatocyte, and an amount ofthe component remaining in other than the bile canaliculus of thehepatocyte.

<Configuration 19>

The method of componential analysis according to Configuration 18,wherein the pretreatment buffer solution and the first buffer solutionare the same buffer solution.

<Configuration 20>

The method of analyzing a drug component according to Configuration 18comprising: prior to the temperature controlling step, a pretreatmenttemperature controlling step of controlling a temperature in at leastone container of the plurality of containers when the at least onecontainer contains the pretreatment buffer solution to be lower than thefirst temperature.

We claim:
 1. A componential analysis method for evaluating an amount ofa chemical substance which is taken up into a cell or excreted from acell, the method comprising: a first step of adding a first buffersolution to a first container and a second container each comprising acell containing a chemical substance to be evaluated, adjusting atemperature within the first container and the second container to afirst temperature and incubating, and adjusting the temperature withinthe first container and the second container to a second temperaturewhich is lower than the first temperature; a second step of collectingthe first buffer solution from each of the first container and thesecond container; a third step of adding a second buffer solutioncomprising a reagent for removing cell-cell adhesion to the firstcontainer after the second step, and adjusting the temperature withinthe first container to a third temperature and incubating; a fourth stepof adding a third buffer solution to the second container after thesecond step, and adjusting the temperature within the second containerto the third temperature and incubating; a first determination step ofdetermining an amount S of the chemical substance which is excreted fromthe cell to the first buffer solution in the first container and/or thesecond container after the first step; a second determination step ofdetermining an amount B1 of the chemical substance which is excretedfrom the cell to the second buffer solution in the first container afterthe third step; a third determination step of determining an amount B2of the chemical substance which is excreted from the cell to the thirdbuffer solution in the second container after the fourth step; andevaluating an amount of the chemical substance excreted from abasal/basolateral side of the cell based on the amount S of the chemicalsubstance.
 2. The componential analysis method of claim 1, wherein themethod comprises: evaluating the amount of the chemical substanceexcreted from an apical side of the cell based on the amount B1 and theamount B2 of the chemical substance.
 3. A componential analysis methodfor evaluating an amount of a chemical substance which is taken up intoa cell or excreted from a cell, the method comprising: a first step ofadding a first buffer solution to a first container and a secondcontainer each comprising a cell containing a chemical substance to beevaluated, adjusting a temperature within the first container and thesecond container to a first temperature and incubating, and adjustingthe temperature within the first container and the second container to asecond temperature which is lower than the first temperature; a secondstep of collecting the first buffer solution from each of the firstcontainer and the second container; a third step of adding a secondbuffer solution to the first container after the second step, andadjusting the temperature within the first container to a thirdtemperature and incubating; a fourth step of adding the second buffersolution to the second container after the second step, and adjustingthe temperature within the second container to a fourth temperaturewhich is lower than the third temperature and incubating; a firstdetermination step of determining an amount B2 of the chemical substancewhich is excreted from the cell to the second buffer solution in thefirst container after the third step; and a second determination step ofdetermining an amount B3 of the chemical substance which is excretedfrom the cell to the second buffer solution in the second containerafter the fourth step.
 4. The componential analysis method of claim 3,wherein the method comprises: a third determination step of determiningan amount S of the chemical substance which is excreted from the cell tothe first buffer solution in the first container and/or the secondcontainer after the first step; and evaluating an amount of the chemicalsubstance excreted from a basal/basolateral side of the cell based onthe amount S of the chemical substance.
 5. The componential analysismethod of claim 3, wherein the method comprises: evaluating an amount ofthe chemical substance excreted from a basal/basolateral side of thecell by diffusion based on the amount B3 of the chemical substance; andevaluating the amount of the chemical substance excreted from thebasal/basolateral side of the cell via transporters based on the amountB2 and the amount B3 of the chemical substance.
 6. A componentialanalysis method for evaluating an amount of a chemical substance whichis taken up into a cell or excreted from a cell, the method comprising:a first step of adding a first buffer solution to a container whichcomprises a cell containing a chemical substance to be evaluated,adjusting a temperature within the container to a first temperature andincubating, and adjusting the temperature within the container to asecond temperature which is lower than the first temperature; a secondstep of collecting the first buffer solution from the container afterthe first step; a third step of adding a second buffer solution to thecontainer after the second step, and adjusting the temperature withinthe container to a third temperature and incubating; a firstdetermination step of determining an amount S of the chemical substancewhich is excreted from the cell to the first buffer solution in thecontainer after the first step; a second determination step ofdetermining an amount B2 of the chemical substance which is excretedfrom the cell to the second buffer solution in the container after thethird step; and evaluating an amount of the chemical substance excretedfrom a basal/basolateral side of the cell based on the amount S of thechemical substance.
 7. The componential analysis method of claim 6,wherein the method comprises: evaluating the amount of the chemicalsubstance excreted from the basal/basolateral side of the cell based onthe amount B2 of the chemical substance.
 8. A componential analysismethod for evaluating an amount of a chemical substance which is takenup into a cell or excreted from a cell, the method comprising: a firststep of adding a first buffer solution to a first container, a secondcontainer and a third container each comprising a cell containing achemical substance to be evaluated, adjusting a temperature within thefirst container, the second container and the third container to a firsttemperature and incubating, and adjusting the temperature within thefirst container, the second container and the third container to asecond temperature which is lower than the first temperature; a secondstep of collecting the first buffer solution from each of the firstcontainer, the second container and the third container; a third step ofadding a second buffer solution comprising a reagent for removingcell-cell adhesion to the first container after the second step, andadjusting the temperature within the first container to a thirdtemperature and incubating; a fourth step of adding a third buffersolution to the second container after the second step, and adjustingthe temperature within the second container to the third temperature andincubating; a fifth step of adding the third buffer solution to thethird container after the second step, and adjusting the temperaturewithin the third container to a fourth temperature which is lower thanthe third temperature and incubating; a first determination step ofdetermining an amount B1 of the chemical substance which is excretedfrom the cell to the second buffer solution in the first container afterthe third step; a second determination step of determining an amount B2of the chemical substance which is excreted from the cell to the thirdbuffer solution in the second container after the fourth step; and athird determination step of determining an amount B3 of the chemicalsubstance which is excreted from the cell to the third buffer solutionin the third container after the fifth step.
 9. The componentialanalysis method of claim 8, wherein the method comprises: a fourthdetermination step of determining an amount S of the chemical substancewhich is excreted from the cell to the first buffer solution in at leastone of the first container, the second container, and the thirdcontainer after the first step; and evaluating an amount of the chemicalsubstance excreted from a basal/basolateral side of the cell based onthe amount S of the chemical substance.
 10. The componential analysismethod of claim 8, wherein the method comprises: evaluating an amount ofthe chemical substance excreted from an apical side of the cell based onthe amount B1 and the amount B2 of the chemical substance; evaluating anamount of the chemical substance excreted from a basal/basolateral sideof the cell by diffusion based on the amount B3 of the chemicalsubstance; and evaluating the amount of the chemical substance excretedfrom the basal/basolateral side of the cell via transporters based onthe amount B2 and the amount B3 of the chemical substance.
 11. Acomponential analysis method for evaluating an amount of a chemicalsubstance which is taken up into a cell or excreted from a cell, themethod comprising: a first step of adding a first buffer solution to afirst container, a second container, a third container and a fourthcontainer each comprising a cell containing a chemical substance to beevaluated, adjusting a temperature within the first container, thesecond container, the third container and the fourth container to afirst temperature and incubating, and adjusting the temperature withinthe first container, the second container, the third container and thefourth container to a second temperature which is lower than the firsttemperature; a second step of collecting the first buffer solution fromeach of the first container, the second container, the third containerand the fourth container; a third step of adding a second buffersolution comprising a reagent for removing cell-cell adhesion to thefirst container after the second step, and adjusting the temperaturewithin the first container to a third temperature and incubating; afourth step of adding a third buffer solution to the second containerafter the second step, and adjusting the temperature within the secondcontainer to the third temperature and incubating; a fifth step ofadding the third buffer solution to the third container after the secondstep, and adjusting the temperature within the third container to afourth temperature which is lower than the third temperature andincubating; a sixth step of adding the second buffer solution comprisinga reagent for removing cell-cell adhesion to the fourth container afterthe second step, and adjusting the temperature within the fourthcontainer to the fourth temperature which is lower than the thirdtemperature and incubating; a first determination step of determining anamount B1 of the chemical substance which is excreted from the cell tothe second buffer solution in the first container after the third step;a second determination step of determining an amount B2 of the chemicalsubstance which is excreted from the cell to the third buffer solutionin the second container after the fourth step; a third determinationstep of determining an amount B3 of the chemical substance which isexcreted from the cell to the third buffer solution in the thirdcontainer after the fifth step; and a fourth determination step ofdetermining an amount B4 of the chemical substance which is excretedfrom the cell to the second buffer solution in the fourth containerafter the sixth step.
 12. The componential analysis method of claim 11,wherein the method comprises: a fifth determination step of determiningan amount S of the chemical substance which is excreted from the cell tothe first buffer solution in at least one of the first container, thesecond container, the third container, and the fourth container afterthe first step; and evaluating an amount of the chemical substanceexcreted from a basal/basolateral side of the cell based on the amount Sof the chemical substance.
 13. The componential analysis method of claim11, wherein the method comprises: evaluating an amount of the chemicalsubstance excreted from a basal/basolateral side of the cell bydiffusion based on the amount B3 of the chemical substance; evaluatingthe amount of the chemical substance excreted from the basal/basolateralside of the cell via transporters based on the amount B2 and the amountB3 of the chemical substance; evaluating an amount of the chemicalsubstance excreted from an apical side of the cell by diffusion based onthe amount B3 and the amount B4 of the chemical substance; andevaluating the amount of the chemical substance excreted from the apicalside of the cell via transporters based on the amount B1, the amount B2,the amount B3 and the amount B4 of the chemical substance.